Project Summary This R01 proposal was written in response to PA-16-233 ?Prescription Drug Abuse?. Prescription opioid abuse is a major public health concern for both men and women. Although opioid addiction has historically exhibited a substantially higher prevalence in men, the gender gap is closing, underscoring the need to conduct research in both sexes. The opioid withdrawal syndrome, characterized by an acute physical syndrome and a long- lasting affective syndrome that includes anhedonia, anxiety, and drug cravings, is a major factor in escalation of opioid use and relapse. Cumulative work from our laboratory and others suggests that opioid withdrawal increases glutamate-mediated activation of the nucleus accumbens shell (NASh), which contributes to the withdrawal syndrome. For example, we have shown that morphine dependence is associated with increased neuronal surface expression of the AMPA glutamate receptor (AMPAR) GluA1 subunit in the NASh, and withdrawal-induced depressive-like states require NASh GluA1 AMPAR activation. It has also been shown that activation of glutamatergic projections from the paraventricular nucleus of the thalamus (PVT) to the NASh is required for expression of morphine withdrawal signs. However, this prior research has been conducted primarily in male rodents that experience withdrawal after experimenter-administered opioids. The regulation and role of NASh glutamatergic signaling in withdrawal from self-administered opioids in males and females is not known. The objective of this proposal is to build on these findings and examine how projection-specific (PVT to NASh) AMPAR-mediated signaling contributes to negative affective states triggered by withdrawal from oxycodone self-administration (SA) in male and female rats. Our central hypothesis is that oxycodone SA triggers projection-specific increases in AMPAR-mediated signaling in the NASh necessary for expression of negative affective states and relapse. We will address this hypothesis in 4 aims in which male and female rats will be exposed to long-access (LgA) oxycodone SA for 2 weeks followed by 2 weeks of abstinence. In Aims 1 and 2 we will examine how withdrawal from LgA oxycodone affects extracellular glutamate levels and synaptic transmission in the NASh using microdialysis (Aim 1) and slice electrophysiology paired with optogenetic activation of PVT to NASh projections (Aim 2). In Aims 3 and 4 we will determine if glutamate release and activation of AMPARs in the NASh is necessary for oxycodone withdrawal-induced negative affective states, as measured with intracranial self-stimulation. We will use chemogenetic modulation of PVT to NASh projections to regulate glutamate release (Aim 3) and viral vector-mediated expression of GluA1ct, which has been shown to block activity-dependent trafficking of GluA1 subunits, to regulate AMPAR transmission in the NASh (Aim 4). Data from these studies will establish a mechanistic link between projection-specific glutamatergic signaling in the NASh and oxycodone withdrawal-induced negative affective states in both males and females, which may ultimately enable development of gender-optimized opioid addiction treatments.